PLEKHG5 deficiency leads to an intermediate form of autosomal-recessive Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease (CMT) comprises a clinically and genetically heterogeneous group of peripheral neuropathies characterized by progressive distal muscle weakness and atrophy, foot deformities and distal sensory loss. Following the analysis of two consanguineous families affected by a medium to late-onset recessive form of intermediate CMT, we identified overlapping regions of homozygosity on chromosome 1p36 with a combined maximum LOD score of 5.4. Molecular investigation of the genes from this region allowed identification of two homozygous mutations in PLEKHG5 that produce premature stop codons and are predicted to result in functional null alleles. Analysis of Plekhg5 in the mouse revealed that this gene is expressed in neurons and glial cells of the peripheral nervous system, and that knockout mice display reduced nerve conduction velocities that are comparable with those of affected individuals from both families. Interestingly, a homozygous PLEKHG5 missense mutation was previously reported in a recessive form of severe childhood onset lower motor neuron disease (LMND) leading to loss of the ability to walk and need for respiratory assistance. Together, these observations indicate that different mutations in PLEKHG5 lead to clinically diverse outcomes (intermediate CMT or LMND) affecting the function of neurons and glial cell

Genome-wide expression profiling and functional characterization of SCA28 lymphoblastoid cell lines reveal impairment in cell growth and activation of apoptotic pathways

BACKGROUND: SCA28 is an autosomal dominant ataxia associated with AFG3L2 gene mutations. We performed a whole genome expression profiling using lymphoblastoid cell lines (LCLs) from four SCA28 patients and six unrelated healthy controls matched for sex and age. METHODS: Gene expression was evaluated with the Affymetrix GeneChip Human Genome U133A 2.0 Arrays and data were validated by real-time PCR. RESULTS: We found 66 genes whose expression was statistically different in SCA28 LCLs, 35 of which were up-regulated and 31 down-regulated. The differentially expressed genes were clustered in five functional categories: (1) regulation of cell proliferation; (2) regulation of programmed cell death; (3) response to oxidative stress; (4) cell adhesion, and (5) chemical homeostasis. To validate these data, we performed functional experiments that proved an impaired SCA28 LCLs growth compared to controls (p\u2009<\u20090.005), an increased number of cells in the G0/G1 phase (p\u2009<\u20090.001), and an increased mortality because of apoptosis (p\u2009<\u20090.05). We also showed that respiratory chain activity and reactive oxygen species levels was not altered, although lipid peroxidation in SCA28 LCLs was increased in basal conditions (p\u2009<\u20090.05). We did not detect mitochondrial DNA large deletions. An increase of TFAM, a crucial protein for mtDNA maintenance, and of DRP1, a key regulator of mitochondrial dynamic mechanism, suggested an alteration of fission/fusion pathways. CONCLUSIONS: Whole genome expression profiling, performed on SCA28 LCLs, allowed us to identify five altered functional categories that characterize the SCA28 LCLs phenotype, the first reported in human cells to our knowledge. \ua9 2013 Mancini et al.; licensee BioMed Central Ltd

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

CHARACTERIZATION OF NANOSTRUCTURED ADSORBENT MATERIALS FOR ENVIRONMENTAL APPLICATIONS

Environmental pollution has grown to become a problem affecting air, water, soil and ecosystems, mainly due to the growth of the world population in combination with rapid economic development, which has led to a massive increase of global production. Indeed, it is linked to the increased use and complexity of chemicals in many human activities. In particular, the global aquatic environment has been affected by widely spread anthropogenic chemicals and, among these, contaminants of emerging interest (CEC), including pharmaceuticals and personal care products, are of particular relevance as they can have a impact on aquatic life and human health. CEC is a term used to describe synthetic or naturally occurring chemicals or any microorganisms that are not commonly monitored in the environment but have the potential to enter the environment and cause known or suspected adverse effects. The main groups of CEC are pharmaceuticals, personal care products, endocrine disruptors, surfactants, persistent organic contaminants, industrial additives and artificial sweeteners. These contaminants are difficult to trace due to the need for specific detection methods, raising the question of how long these contaminants are prevalent in the ecosystem and how the contamination process can be reversed or reduced. Furthermore, several CECs are unlikely to be removed from conventional wastewater treatment (WWT) processes. Advanced wastewater treatment technologies have been identified to be effective in treating contaminated water, such as nanofiltration, reverse osmosis, ozonation and chemical oxidation. Among the different solutions, adsorption, an established technology, is still considered a reliable and robust method to purify aqueous solutions at low cost and with high efficiency. One of the main advantages of adsorption-based technologies is that they are capable of removing contaminants in very low concentration ranges, an operating condition in which most other separation techniques are poorly efficient due to the small concentration gradients involved. . Furthermore, adsorption is a versatile method that can remove many different organic and inorganic compounds at the same time, provided a suitable blend of adsorbent materials is employed.This thesis focused on evaluating different strategies for removing pollutants from the aqueous matrix. In particular, we have studied methods based on adsorption, and in this case we have selected adsorbent materials different from each other in terms of structure, chemical composition and operating conditions, which can be divided into two classes: (i) microporous silica aluminate adsorbent (zeolites) and (ii) carbon-based mixed matrix (MMM) membranes. In this thesis the adsorbing properties of zeolites towards different classes of CEC (drugs, PFAS and organic contaminants) in aqueous solutions were studied in order to study the efficiency of these siliceous materials in two applications, namely: (1) the removal of contaminants from aqueous matrix, (2) the pre-concentration phase for the analysis of micropollutants. Furthermore, the properties of MMM adsorption towards perfluorinated compounds were also investigated. MMMs are composed of a continuous polymeric phase and a dispersed inorganic filler. This thesis includes a study on advanced oxidation processes for the degradation of pharmaceutical products in an aqueous environment. In particular, two different photocatalysts have been studied: (i) sodium decatungstate and (ii) tungsten trioxide, these semiconductors have the common ability to photoproduce .OH radicals in aqueous solutions.L'inquinamento ambientale è cresciuto fino a diventare un problema che colpisce l'aria, l'acqua, il suolo e gli ecosistemi, principalmente a causa della crescita della popolazione mondiale in combinazione con un rapido sviluppo economico, che ha portato a un massiccio aumento della produzione globale. In effetti, è legato all'aumento dell'utilizzo e della complessità delle sostanze chimiche in molte attività umane. In particolare, l'ambiente acquatico globale è stato influenzato da sostanze chimiche antropogeniche ampiamente diffuse e, tra queste, i contaminanti di interesse emergente (CEC), compresi i prodotti farmaceutici e i prodotti per la cura personale, sono di particolare rilevanza poiché possono avere un impatto sulla vita acquatica e salute umana. CEC è un termine usato per descrivere sostanze chimiche sintetiche o presenti in natura o qualsiasi microorganismo che non è comunemente monitorato nell'ambiente ma ha il potenziale di entrare nell'ambiente e causare effetti negativi noti o sospetti. I principali gruppi di CEC sono prodotti farmaceutici, prodotti per la cura personale, interferenti endocrini, tensioattivi, contaminanti organici persistenti, additivi industriali e dolcificanti artificiali. Questi contaminanti sono difficili da rintracciare a causa della necessità di metodi di rilevamento specifici, sollevando la domanda su quanto tempo questi contaminanti siano prevalenti ell'ecosistema e come il processo di contaminazione possa essere invertito o ridotto. Inoltre, è improbabile che diversi CEC vengano rimossi dai processi convenzionali di trattamento delle acque reflue (WWT). Le tecnologie avanzate di trattamento delle acque reflue sono state identificate per essere efficaci nel trattamento delle acque contaminate, come la nanofiltrazione, l'osmosi inversa,l'ozonizzazione e l'ossidazione chimica. Tra le diverse soluzioni, l'adsorbimento, una tecnologia consolidata, è ancora considerata un metodo affidabile e robusto per purificare soluzioni acquose a basso costo e con alta efficienza. Uno dei principali vantaggi delle tecnologie basate sull'adsorbimento è che sono in grado di rimuovere i contaminanti in intervalli di concentrazione molto bassi, una condizione operativa in cui la maggior parte delle altre tecniche di separazione sono scarsamente efficienti a causa dei piccoli gradienti di concentrazione coinvolti. Inoltre, l'adsorbimento è un metodo versatile che può rimuovere contemporaneamente molti diversi composti organici e inorganici, a condizione che venga impiegata una miscela adatta di materiali adsorbenti.Questa tesi si è concentrata sulla valutazione di diverse strategie per rimuovere gli inquinanti dalla matrice acquosa. In particolare, abbiamo studiato metodi basati sull'adsorbimento, e in questo caso sono stati selezionati materiali adsorbenti diversi tra loro per struttura, composizione chimica e condizioni operative, che possono essere suddivisi in due classi: (i) adsorbente microporoso silico alluminato (zeoliti) e (ii) membrane a matrice mista a base di carbonio (MMM). In questa tesi sono state studiate le proprietà adsorbenti delle zeoliti verso diverse classi di CEC (farmaci, PFAS e contaminanti organici) in soluzioni acquose al fine di studiare l'efficienza di questi materiali silicei in due applicazioni, ovvero: (1) la rimozione di contaminanti da matrice acquosa, (2) la fase di pre-concentrazione per l'analisi dei microinquinanti. Inoltre, sono state studiate anche le proprietà di adsorbimento MMM verso i composti perfluorurati. Le MMM sono composti da una fase polimerica continua e da una carica inorganica dispersa. Questa tesi include uno studio sui processi di ossidazione avanzati per la degradazione di prodotti farmaceutici in ambiente acquoso. In particolare sono stati studiati due diversi fotocatalizzatori: (i) decatungstato di sodio e (ii) triossido di tungsteno, questi semiconduttori hanno la capacità comune di fotoprodurre radicali .OH in soluzioni acquose

An advanced oxidation process by photoexcited heterogeneous sodium decatungstate for the degradation of drugs present in aqueous environment

The ability of decatungstate anion (W10O324-) to photoproduce OH center dot radicals from water is retained when the polyoxoanion is immobilized on solid supports and can be exploited as a heterogeneous photocatalytic process aimed to water remediation. However, we show that interaction between OH center dot radicals and drug molecules depends on the physical chemical properties of the chosen support. In fact, we demonstrate that silica particles covered by decatungstate anions are not efficient in the degradation of carbamazepine and sulfamethoxazole (ubiquitary contaminants of natural waters) that are respectively neutral and negatively charged at operating pH of 6. On the contrary, entrapment of W10O324- inside a mesoporous organosilica matrix leads to a heterogeneous photocatalytic system with proper characteristic of hydrophobicity. Organic molecules enter mesopores and easily leave the aqueous environment. Inside pores, the reaction between photoproduced OH center dot radicals is favored. Mono- and di-hydroxylated products precede fragmentation and degradation of the investigated drug

Heterogeneous sodium decatungstate catalysts for contaminants degradation in aqueous matrix

The ability of decatungstate to photoproduce •OH radicals from water is retained when the polyoxoanion is immobilized on solid supports and can be exploited as a heterogeneous photocatalytic system for advanced oxidation process in water remediation field. Herein, we show that interaction between •OH radicals and drug molecules depends on the physical chemical properties of the chosen support. In particular, it depends on the charge of the molecule at operating pH of 6

Anticancer Activity of Aqueous Extracts from Asparagus officinalis L. Byproduct on Breast Cancer Cells

Cultivation of asparagus (Asparagus officinalis L.; Asp) for food and medicinal use has taken place since the early Roman Empire. Today, Asp represents a worldwide diffuse perennial crop. Lower portions of the spears represent a food industry waste product that can be used to extract bioactive molecules. In this study, aqueous extracts derived from the non-edible portion of the plant (hard stem) were prepared and characterized for chemical content. Furthermore, the biocompatibility and bioactivity of Asp aqueous extracts were assessed in vitro on normal fibroblasts and on breast cancer cell lines. Results showed no interference with fibroblast viability, while a remarkable cytostatic concentration-dependent activity, with significant G1/S cell cycle arrest, was specifically observed in breast cancer cells without apoptosis induction. Asp extracts were also shown to significantly inhibit cell migration. Further analyses showed that Asp extracts were characterized by specific pro-oxidant activity against tumoral cells, and, importantly, that their combination with menadione resulted in a significant enhancement of oxidants production with respect to menadione alone in breast cancer cells but not in normal cells. This selectivity of action on tumoral cells, together with the easiness of their preparation, makes the aqueous Asp extracts very attractive for further investigation in breast cancer research, particularly to investigate their role as possible co-adjuvant agents of clinical drug therapies

Insights into adsorption of chlorobenzene in high silica MFI and FAU zeolites gained from chromatographic and diffractometric techniques

In this work, the capability of two commercial high silica zeolites (HSZs), namely ZSM-5 and Y, for the removal of chlorobenzene (CB) from water was investigated by combining chromatographic and diffractometric techniques. The adsorption isotherms and kinetics of CB on ZSM-5 and Y zeolites were determined from batch tests. The adsorption kinetics were very fast; the time to reach equilibrium was less than 10 min. The equilibrium data of CB on the two HSZs showed dissimilarities that are particularly evident in the adsorption data concerning the low concentration range, where Y zeolite is characterized by low adsorption. On the contrary, at higher solution concentrations the adsorption capacity of Y is higher than that of ZSM-5. The crystalline structures of Y and ZSM-5 saturated with CB were investigated by X-ray diffraction (XRD) techniques. Rietveld refinement analyses of XRD data allowed for quantitative probing of the structural modifications of both zeolites after CB adsorption and provided insight into the preferred zeolite adsorption sites in both microporous materials. The refined frameworkâ\u80\u93extraframework bond distances confirm that interactions between the selected organic contaminant and hydrophobic zeolites are mediated via co-adsorbed H2O. The occurrence of H2Oâ\u80\u93CBâ\u80\u93framework oxygen oligomers explains variations in both the unit cell parameters and the shape of the channels, clearly confirming that water plays a very relevant role in controlling the diffusion and adsorption processes in hydrophobic zeolites

Occurance of heavy metals in sediments and biota in Northern Adriatic Sea

Due to the low depth and the modest water exchange, Adriatic Sea is considered a fragile area. In particular, the Northern area receives fresh water input from a series of sources; the most important one is the Po River which drains intensely cultivated and industrialized hinterland areas and releases into the sea great amounts of dissolved and particulate materials, including anthropogenic contaminants. The aim of this study was to monitor heavy metals concentrations, in biotic and abiotic samples. Heavy metals, especially mercury, are often present in industrial and municipal effluents and are ubiquitary pollutants frequently detected in the environment that can cause long-term effects on marine ecosystems. In particular, samples of sediments, fishes and filter-feeding organisms, collected in the North-Est area of Adriatic Sea were analyzed. The samples were homogenized and digested with nitric acid and hydrogen peroxide by using microwave-oven technique. The analyses were performed by graphite furnace absorption spectrometry (Cd and As), atomic emission spectrometry (ICP-AES) (Ba, Cr, Fe, Mn, Ni, Pb, Cu and Zn) and cold vapour atomic absorption spectrometry after amalgamation traps on gold for the preconcentration of mercury in order to lower the detection limit of the analyte